TY - JOUR
T1 - Human NOTCH4 is a key target of RUNX1 in megakaryocytic differentiation
AU - Li, Yueying
AU - Jin, Chen
AU - Bai, Hao
AU - Gao, Yongxing
AU - Sun, Shu
AU - Chen, Lei
AU - Qin, Lei
AU - Liu, Paul P.
AU - Cheng, Linzhao
AU - Wang, Qian Fei
N1 - Funding Information:
This work was supported by grants from the External Cooperation Program of Bureau of International Co-operation Chinese Academy of Sciences (153F11KYSB20150013) (Q.-F.W.), the National Natural Science Foundation of China (81770109) (Y.L.), National Institutes of Health, National Heart, Lung, and Blood Institute grant R01-HL130676 (L.C.), and the Youth Innovation Promotion Association of Chinese Academy of Sciences (Y.L.). L.C. is also supported by Edythe Harris Lucas and Clara Lucas Lynn Chair in Hematology of Johns Hopkins University. P.L. is supported by the Intramural Research Program, National Human Genome Research Institute, National Institutes of Health. This work was also supported by grant from the K. C. Wong Education Foundation.
PY - 2018/1/11
Y1 - 2018/1/11
N2 - Megakaryocytes (MKs) in adult marrow produce platelets that play important roles in blood coagulation and hemostasis. Monoallelic mutations of the master transcription factor gene RUNX1 lead to familial platelet disorder (FPD) characterized by defective MK and platelet development. However, the molecular mechanisms of FPD remain unclear. Previously, we generated human induced pluripotent stem cells (iPSCs) from patients with FPD containing a RUNX1 nonsense mutation. Production of MKs from the FPD-iPSCs was reduced, and targeted correction of the RUNX1 mutation restored MK production. In this study, we used isogenic pairs of FPD-iPSCs and the MK differentiation system to identify RUNX1 target genes. Using integrative genomic analysis of hematopoietic progenitor cells generated from FPD-iPSCs, and mutation-corrected isogenic controls, we identified 2 gene sets the transcription of which is either up- or downregulated by RUNX1 in mutationcorrected iPSCs. Notably, NOTCH4 expression was negatively controlled by RUNX1 via a novel regulatory DNA element within the locus, and we examined its involvement in MK generation. Specific inactivation of NOTCH4 by an improved CRISPR-Cas9 system in human iPSCs enhanced megakaryopoiesis. Moreover, small molecules known to inhibit Notch signaling promoted MK generation from both normal human iPSCs and postnatal CD341 hematopoietic stem and progenitor cells. Our study newly identified NOTCH4 as a RUNX1 target gene and revealed a previously unappreciated role of NOTCH4 signaling in promoting human megakaryopoiesis. Our work suggests that human iPSCs with monogenic mutations have the potential to serve as an invaluable resource for discovery of novel druggable targets.
AB - Megakaryocytes (MKs) in adult marrow produce platelets that play important roles in blood coagulation and hemostasis. Monoallelic mutations of the master transcription factor gene RUNX1 lead to familial platelet disorder (FPD) characterized by defective MK and platelet development. However, the molecular mechanisms of FPD remain unclear. Previously, we generated human induced pluripotent stem cells (iPSCs) from patients with FPD containing a RUNX1 nonsense mutation. Production of MKs from the FPD-iPSCs was reduced, and targeted correction of the RUNX1 mutation restored MK production. In this study, we used isogenic pairs of FPD-iPSCs and the MK differentiation system to identify RUNX1 target genes. Using integrative genomic analysis of hematopoietic progenitor cells generated from FPD-iPSCs, and mutation-corrected isogenic controls, we identified 2 gene sets the transcription of which is either up- or downregulated by RUNX1 in mutationcorrected iPSCs. Notably, NOTCH4 expression was negatively controlled by RUNX1 via a novel regulatory DNA element within the locus, and we examined its involvement in MK generation. Specific inactivation of NOTCH4 by an improved CRISPR-Cas9 system in human iPSCs enhanced megakaryopoiesis. Moreover, small molecules known to inhibit Notch signaling promoted MK generation from both normal human iPSCs and postnatal CD341 hematopoietic stem and progenitor cells. Our study newly identified NOTCH4 as a RUNX1 target gene and revealed a previously unappreciated role of NOTCH4 signaling in promoting human megakaryopoiesis. Our work suggests that human iPSCs with monogenic mutations have the potential to serve as an invaluable resource for discovery of novel druggable targets.
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U2 - 10.1182/blood-2017-04-780379
DO - 10.1182/blood-2017-04-780379
M3 - Article
C2 - 29101237
AN - SCOPUS:85040466081
SN - 0006-4971
VL - 131
SP - 191
EP - 201
JO - Blood
JF - Blood
IS - 2
ER -